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R Makino

Publications and source records attributed to R Makino.

At least 37 records · Page 2Linked to original sources
PubMed1996

The 5' end sequences and exon organization in rat regucalcin gene.

The 5'-flanking region of the gene for a Ca(2+)-binding protein regucalcin was cloned from a rat genomic library which was constructed in lambda EMBL3 SP6/T7 vector. The genomic library was screened by using the radiolabeled probe with the 5' region (0.5 kb) of rat regucalcin complementary deoxyribonucleic acid (cDNA). Positive clone had the 5.5 kb fragment which was hybridized with the 5'-probe. This fragment contained three exons (I-III) of the gene coding for a rat regucalcin. The nucleotide sequence of exons completely agreed with that of a rat regucalcin cDNA clone. A supposed translational initiation site existed in the exon II. Homology analysis showed that a putative transcription start site in the rat regucalcin gene was located at position 26 downstream from a TATA-box. Another upstream element, a CCAAT box-like sequence, was located at -170. Moreover, there were many regulatory elements (Hox, AP-1, AP-2 and AP-4) in the 5'-flanking region of the rat regucalcin gene. The organization of rat regucalcin gene seemed to be about 18 kb in size and consisted of seven exons and six introns.

Amino Acid Sequence↗
PubMed1996

Role of Arg112 of cytochrome p450cam in the electron transfer from reduced putidaredoxin. Analyses with site-directed mutants.

The mechanism for the reduction of ferric cytochrome P450cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450cam, in which Arg112, an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residues. The affinity of reduced putidaredoxin for ferric cytochrome P450cam to form a diprotein complex was decreased greatly by changing Arg112 to a neutral amino acid such as Cys, Met, or Tyr. The rate of intracomplex electron transfer from putidaredoxin to cytochrome P450cam also diminished upon replacing the basic residue with neutral ones, being 42, 18, 4.0, 1.3, and 0. 16 s-1 for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. Furthermore, the oxidation-reduction potential of cytochrome P450cam (Fe3+/Fe2+ couple) decreased in a similar way to the decrease in the rate of electron transfer upon amino acid substitution; the values were -138, -162, -182, -200, and -195 mV for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. These results indicate that the amino acid substitution at position 112 affects the oxidation-reduction potential of the heme iron in cytochrome P450cam, thereby diminishing the rate of electron transfer between the two metal centers. The rate of electron transfer from putidaredoxin to oxyferrous cytochrome P450cam also diminished upon substitution of Arg112 with a neutral amino acid.

Arginine↗
PubMed1996

The structure-function relationship and reduction potentials of high oxidation states of myoglobin and peroxidase.

In these studies, we substitute electron-withdrawing (diacetyl) or -donating (diethyl) groups at the 2- and 4-positions of the heme in sperm whale Mb and HRP, and examine the structural and biochemical consequences. X-ray absorption spectroscopy shows that increased electron density at the heme results in an increased iron-pyrrole nitrogen average distance in both HRP and Mb, while decreased electron density results in shorter average distances. In HRP, the proximal ligand is constrained by a H-bonding network, and axial effects are manifested entirely at the distal site. Conversely, in Mb, where the proximal ligand is less constrained, axial effects are seen at the proximal side. In HRP, electron density at the heme iron depends linearly on pK3, a measure of the basicity of the porphyrin pyrrole nitrogens [Yamada, H., Makino, R., & Yamazaki, I. (1975) Arch. Biochem. Biophys. 169, 344-353]. Using diethyl substitution (pK3 = 5.8) and diacetyl substitution (pK3 = 3.3) in HRP and Mb, we measured the one-electron reduction potentials (E(O)') of HRP compounds I and II and ferryl Mb. Compound I showed a decreased E(O)' with increasing electron density at the heme (pK3), similar to E(O)' of ferric HRP. E(O)' of HRP compound II and ferryl Mb showed an opposite dependence. This behavior of E(O)', while initially surprising, can be explained by the apparent net positive charge on the iron porphyrin in each oxidation state of the hemoproteins.

Animals↗
PubMed1996

Expression of calcium-binding protein regucalcin mRNA in hepatoma cells.

Whether the gene expression of hepatic Ca(2+)-binding protein regucalcin is altered in hepatomas was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). Rat hepatoma was induced by continuous feeding of basal diet containing 0.06% 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB). After 35 weeks feeding, rats were sacrificed, and the non-tumorous and tumorous tissues of the livers were removed. In individual rats, the regucalcin mRNA levels in the tumorous tissues were generally decreased in comparison with that of the non-tumorous tissues of the chemical-fed rats, although the chemical administration might decrease the mRNA expression in normal rat liver, suggesting that the chemical administration causes a suppressive effect on the mRNA expression. When the genomic DNA extracted from the liver tumorous tissues was digested with restriction enzymes (EcoRI, BamHI and HindIII) and analyzed by Southern blotting, no rear-ranged band was found in the regucalcin gene from the hepatoma. Interestingly, in the transplantable Morris hepatoma cells, the regucalcin mRNA was markedly expressed, while the albumin mRNA was expressed only slightly. The present study demonstrates that regucalcin mRNA is clearly expressed in the transformed cells (Morris hepatoma cells).

Animals↗
PubMed1995

Iron-ligand structure and iron redox property of nitric oxide reductase cytochrome P450nor from Fusarium oxysporum: relevance to its NO reduction activity.

We studied the nitric oxide reductase, cytochrome P450nor, purified from a denitrifying fungus Fusarium oxysporum with electron paramagnetic resonance spectral and redox potential measurements. The EPR spectral features of P450nor in the ferric resting, the ferric cyanide-bound, and the ferrous NO-bound forms were the same as the corresponding ones of other general P450s such as Pseudomonas putida P450cam. In contrast, the metyrapone complex of ferric P450nor gave an EPR spectrum with significantly different g values from that of P450cam. The EPR results were explained in terms of similarity in the immediate configuration of the S(-)-Fe-ligand (H2O, CN-, NO) structure between P450nor and P450cam but a structural difference at the heme distal pocket, especially in the substrate binding domain; P450cam has a camphor binding domain, while P450nor does not. In spite of the same S(-)-Fe-H2O configuration, the redox potential of P450nor in the ferric/ferrous couple was measured to be -307 mV, which is much lower than those of the camphor-bound (-140 mV) and -free (-250 mV) P450cam. The lower redox potential could be attributable to the different electrostatic interaction of the heme with its surroundings; e.g., the heme environment of P450nor is charged either more negatively or less positively than P450cam.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1995

Loss of heterozygosity at 11p15 in malignant glioma.

Deletions of loci on chromosome 11p have been found frequently in several malignant tumors including gliomas, suggesting the presence of tumor suppressor genes. We analyzed 38 gliomas [26 malignant gliomas (grades III and IV) and 12 less malignant gliomas (grade I and II)] for loss of heterozygosity using microsatellite sequences on 11p as polymorphic markers. Loss of heterozygosity was found in 8 of 26 malignant gliomas (31%) but not in the less malignant gliomas. In the region with loss of heterozygosity, loci on 11p15.5-pter were commonly deleted. Our results suggest that a putative tumor suppressor gene involved in malignant progression of gliomas is located in an approximately 21-cM region on 11p15.5-pter.

Base Sequence↗
PubMed1995

Thermodynamic aspects of the CO-binding reaction to cytochrome P-450cam. Relevance with their biological significance and structure.

The CO-binding kinetics of cytochrome P-450cam(+) and P-450cam(-) have been measured in the millisecond time domain using a flash photolysis method. We have determined the reaction coordinates for free energy, enthalpy and entropy from the temperature dependence of the overall rate constants of the bimolecular forward (on) and backward (off) reactions. Comparing the thermodynamic profiles of P-450cam with that of myoglobin (Mb) reported so far, the enthalpy and the entropy coordinates exhibit the following remarkable characteristics. The CO-binding equilibrium: The stability of the CO-complex is perfectly entropy-driven for P-450cam, while enthalpy-driven for Mb. This entropy-driven feature for P-450cam is enhanced by the dissociating d-camphor. The on and off activation processes: The on and off reactions for P-450cam are dominantly controlled by the enthalpy and entropy terms, respectively, while those for Mb are rather the reverse of the case of P-450cam. The dissociation of d-camphor has a significant effect on the on reaction but no effect on the off reaction. Analyzing these thermodynamic features on the basis of the physical chemistry in the solution reaction, it was found that these characteristic profiles arise from the difference in the global structural change between the proteins. Namely, during the equilibrium process of the CO binding, this structural change is accompanied by a larger increase in the degree of freedom in P-450cam than in Mb. We discussed the correlations between the structural changes and their biological significance.

Camphor↗
PubMed1995

Cloning and characterization of rat cellular nucleic acid binding protein (CNBP) cDNA.

We cloned and sequenced the cDNAs which code for rat cellular nucleic acid binding protein (CNBP). In-frame insertion/deletion differences were found among the clones at two sites in the open reading frame, suggesting alternative splicing of the message or the presence of multiple genes which code for this protein. The deduced amino acid sequence revealed that one rat CNBP sequence was completely identical to its human counterpart. This striking conservation, together with the fact that homologous genes have been found in various organisms including Schizosaccharomyces pombe, suggests that CNBP plays a basic biological role in eukaryotic cells. The recombinant GST-CNBP fusion protein produced in Escherichia coli bound to a G-rich single-stranded RNA and DNA in a sequence-specific manner.

Amino Acid Sequence↗
PubMed1994

Cloning and characterization of a c-myc intron binding protein (MIBP1).

The cDNA for a c-myc intron 1 binding protein 1 (MIBP1) in the rat was isolated from lambda gt11 and lambda ZAPII cDNA libraries. Sequencing of the cDNA clones revealed a long ORF which encoded a putative protein of 2437 amino acid residues. This protein has two widely separated zinc finger regions, each of which carries C2H2 motifs. When expressed in E. coli as a fusion protein, part of the MIBP1 showed sequence-specific binding to the target sequence, i.e., a 9-bp sequence in the rat c-myc intron 1. MIBP1 is most likely the rat counterpart of human MHC binding protein-2 (MBP-2/HIV-EP2), based on the 86% similarity in nucleotide sequence and 93% similarity in amno acid sequence. Northern blotting revealed a high level of MIBP1 mRNA in the brain.

Amino Acid Sequence↗
PubMed1994

EPR studies on the photoproducts of ferric cytochrome P450cam (CYP101) nitrosyl complexes: effects of camphor and its analogues on ligand-bound structures.

Photolyzed products of the NO complexes of ferric cytochrome P450cam both in the substrate-free and several substrate-bound states were trapped and examined by EPR spectroscopy at 5 K. In the absence of substrate, the photoproduct exhibited ferric high- and low-spin signals, neither of which showed the line-width broadening characteristic of magnetic interaction between photodissociated NO and the heme iron. This finding indicates that photodissociated NO can diffuse to the unconstrained distal heme pocket giving the high-spin heme, and that a part of the high-spin heme species converts to the low-spin heme upon coordinating an aqua molecule. When a substrate, camphor or adamantanone, was bound at the site above the heme, the photoproduct exhibited widespread EPR absorptions together with a new distinct signal at g approximately 4.4. The new signals are assignable to a weakly spin-coupled species between the ferric heme iron and the photodissociated NO, indicating that the NO molecule is in close proximity to the heme iron by the steric crowding of the bound substrate. The photoproduct of the norcamphor complex exhibited a spin-coupled EPR signal at g approximately 5, in which the coupling is suggested to be weaker than that of the camphor-bound enzyme. On the other hand, the photoproduct of the NO complex in an adamantane-bound state only yielded low-spin signals, and exhibited no spin-coupled signals. This result suggests that adamantane is mobile in the substrate pocket due to the lack of hydrogen bond formation with Tyr96.(ABSTRACT TRUNCATED AT 250 WORDS)

Adamantane↗
PubMed1994

Absence of activating mutations of the RAF1 protooncogene in human lung cancer.

Recently, the RAF protein has been demonstrated to be a direct effector of RAS protein in a RAS-mediated signal transduction pathway. Activations of the RAF1 gene by small mutations, such as point mutations in the kinase domain and a tetrapeptide insertion into conserved region 2, have been suggested from analyses of chemically induced lung cancers in mice and by site-directed mutagenesis. We investigated the presence of small mutations of the RAF1 gene in human lung carcinomas, especially in those not carrying the mutated RAS gene, expecting that aberrations of the RAF1 gene might play a role complementary to RAS gene mutations in tumorigenesis. Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products of DNA samples from 140 patients revealed no tumor specific mutations of the RAF1 gene in any of these specimens. This result suggests that mutations of the RAF1 gene are not involved in tumorigenesis in human lung.

Base Sequence↗
PubMed1993

Essential role of the Arg112 residue of cytochrome P450cam for electron transfer from reduced putidaredoxin.

Cytochrome P450cam (CYP101) of Pseudomonas putida PpG1 in which Arg112 is substituted by Cys was isolated by in vitro random mutagenesis of the camC gene DNA coding for P450cam. The absorption spectra of the purified mutant enzyme were similar to those of the wild type enzyme, but its substrate-dependent NADH oxidation activity in the presence of putidaredoxin (Pd) and putidaredoxin reductase (PdR) was extremely low. The rate constant of electron transfer from reduced Pd to the heme of the mutant P450cam, measured on an anaerobic stopped flow apparatus, was 1/400 of that of the wild type enzyme and the dissociation constant of the mutant P450cam for oxidized Pd was several fold higher than that of the wild type enzyme. A considerable decrease in mid-point potential of the mutant enzyme was also noted. We conclude that Arg112, which is located on the surface of the P450cam molecule and hydrogen-bonded to one of the heme propionate chains, plays an essential role in the electron transfer from Pd.

Arginine↗
PubMed1993

Specific modification of structure and property of myoglobin by the formation of tetrazolylhistidine 64(E7). Reaction of the modified myoglobin with molecular oxygen.

Tetrazole-myoglobin (Tet-Mb), a site selectively modified myoglobin with tetrazole anion (-CN4-) covalently attached to the imidazole N epsilon of the distal histidine 64(E7) (see Fig. 1; Kamiya, N., Shiro, Y., Iwata, T., Iizuka, T., and Iwasaki, H. (1991) J. Am. Chem. Soc. 113, 1826-1829), exhibited unique properties in the reduction from ferric to ferrous states and in the reaction of its deoxy form with O2. The redox potential of Tet-Mb is obtained to be -193 mV, which is much lower than that of unmodified (native) myoglobin (50 mV), possibly due to the electrostatic interaction between the heme iron and the tetrazole group. The ferrous deoxy form of Tet-Mb was rapidly oxidized to its ferric form in the reaction with O2 at room temperature through an intermediary formation of its oxy form and with the generation of O2-. The oxy form of Tet-Mb can be detected by the optical spectral measurement at -12 degrees C, the rapid scan measurement at room temperature, and the electron spin resonance measurement of its cobalt-substituted derivative (Tet-Mb(Co2+)) at 77 K. In the kinetic measurement of the O2 binding reaction to Tet-Mb, its association and dissociation rate constants in the bimolecular reaction were 6.1 x 10(7) M-1 s-1 and 2200 s-1, respectively, showing that the tetrazole modification of His-64 extremely accelerates its association and dissociation rates. Taken together with the extremely fast autoxidation rate (53 h-1) obtained, these kinetic results suggested that the channel of O2 from the solvent region to the protein interior is open enough to pass the external ligand. The structure is discussed in relation to those of some genetic mutants. Taking these properties, we demonstrated that Tet-Mb can catalyze O2 consumption to generate O2-, coupled with the NADH-supported enzymatic reduction system of cytochrome P-450cam under an aerobic condition.

Amino Acid Sequence↗
PubMed1993

Superoxide-producing cytochrome b. Enzymatic and electron paramagnetic resonance properties of cytochrome b558 purified from neutrophils.

Molecular properties of superoxide (O2-)-producing cytochrome b558 purified from neutrophils were investigated focusing on the mechanism of the catalytic reaction. The purified cytochrome, which was depleted of FAD, exhibited high O2(-)-generating activity with consumption of NADPH in the presence of microsomal NADPH-cytochrome P-450 reductase. Exogenous additions of CO, CN-, or N3- had no effect on the enzymatic activity. Potentiometric titration of the ferric-ferrous couple of the cytochrome showed that the midpoint reduction potential was -255 mV at pH 7.4. When the reaction of the reduced cytochrome with O2 was analyzed by stopped flow and rapid scanning spectrophotometry, the ferrous form was found to be converted to the ferric form at a rate constant of 9.3 x 10(6) M-1 s-1 at 10 degrees C without showing formation of an oxygenated intermediate. EPR measurement of the ferric cytochrome at 10 K showed that the electronic spin state was in a low spin with g values of 3.2, 2.05, and 1.5. These results suggest that the heme in a six-coordinated low spin state catalyzes one electron reduction of O2 without ligation of O2 to the heme iron during the catalytic cycle.

Animals↗
PubMed1993

Roles of proximal ligand in heme proteins: replacement of proximal histidine of human myoglobin with cysteine and tyrosine by site-directed mutagenesis as models for P-450, chloroperoxidase, and catalase.

Histidine-93(F8) in human myoglobin (Mb), which is the proximal ligand of the heme iron, has been replaced with cysteine or tyrosine by site-directed mutagenesis. The resultant proximal cysteine and tyrosine mutant Mbs (H93C and H93Y Mbs, respectively) exhibit the altered axial ligation analogous to P-450, chloroperoxidase, and catalase. Coordination of cysteine or tyrosine to the ferric heme iron is confirmed by spectroscopic measurements including electronic absorption, hyperfine-shifted 1H-NMR, EPR, resonance Raman spectroscopies, and redox potential measurements of ferric/ferrous couple. H93C Mb is five-coordinate ferric high-spin with the proximal cysteine. H93Y Mb bearing the proximal tyrosine ligated to the iron is also in a ferric high-spin, five-coordinate state. The reactions of the mutants with cumene hydroperoxide show that the thiolate ligand enhances heterolytic O-O bond cleavage of the oxidant, while the phenolate ligand hardly affects the heterolysis/homolysis ratio for O-O bond scission in comparison with wild-type Mb. Monooxygenase activities such as epoxidation of styrene and N-demethylation of N,N-dimethylaniline, and catalase activity (dismutation of hydrogen peroxide) by wild-type Mb and the mutants, are examined by using H2O2. The increase of the catalytic activities by the mutation was, at most, 5-fold in the epoxidation reaction.

Benzene Derivatives↗
PubMed1992

Observation of the FeIV=O stretching Raman band for a thiolate-ligated heme protein. Compound I of chloroperoxidase.

The FeIV=O stretching vibration has never been identified for a cysteine-coordinated heme enzyme. In this study, resonance Raman and visible absorption spectra were observed simultaneously for transient species in the catalytic reaction of chloroperoxidase with hydrogen peroxide by using our original apparatus for mixed-flow and Raman/absorption simultaneous measurements. For the first intermediate, the FeIV=O stretching Raman band was observed at 790 cm-1, which shifted to 756 cm-1 with the 18O derivative, but the v4 band was too weak to be identified. This suggested the formation of an oxoferryl porphyrin pi cation radical. The second intermediate gave an intense v4 band at 1,372 cm-1 but no oxygen isotope-sensitive Raman band, suggesting oxygen exchange with bulk water.

Chloride Peroxidase↗